Hanahan competent E.coli

Andy Law Big Nose Andy.Law at bbsrc.ac.uk
Wed Apr 19 08:19:44 EST 1995


In article <3n2mqg$3i4 at mserv1.dl.ac.uk>, LOGAND
<logand at msdos.montpellier.inra.fr> wrote:

  > Hi,
  > 
  > I am planning to prepare competent cells by the Hanahan method to use for 
  > the transformation of many ligation reactions (nested deletions prepared 
  > using the Pharmacia kit). I have never used this transformation protocol 
  > before, Sambrook et al. states that the number of cells in the culture 
  > should not exceed 1x10^8/ml and one should determine the relationship 
  > between OD and viable cell number/ml for the strain to be used. Well, I 
  > would love to be able to omit (or at least limit) this time consumming step 
  > - thus, can anyone give me a value for this using DH5a? I realise I will 
  > have to check/re-calculate this due to differences in spectrophotometers 
  > (light scattering etc) but at least I would have an approx. starting point.

We go for about 0.5-0.6 absorbance at 550nm using Hanahan RbCl method and
DH5a cells. It's not as critical as it's made out to be, although you have
to hit it on the up.


  > Finally, do XL1 blue cells give worse or better transformation frequencies?
  > 

Depends on the construct, phase of the moon, time of day, direction of the
wind......
-- 
Andy Law

( Andy.Law @ bbsrc.ac.uk )
( Big Nose in Edinburgh )



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